Project Summary/Abstract Ticks transmit several pathogens including flaviviruses that cause diseases in humans. The molecular determinants and mechanisms of arthropod-borne flavivirus transmission to the vertebrate host are poorly understood. In this study, we provide strong preliminary data that show for the first time that a cell line from medically important arthropods, such as ticks, secretes exosomes that mediate transmission of flavivirus RNA and proteins to the human cells. We noted that tick-borne Langat virus (LGTV), a model pathogen closely related to tick-borne encephalitis virus (TBEV), profusely uses tick exosomes for transmission of viral RNA and proteins to the human- skin keratinocytes and blood endothelial cells. Cryo- EM analysis revealed presence of tick exosomes with the size range of 30 to 200 nm in diameter. Detection of both positive and negative LGTV RNA strands and proteins such as Envelope (E) and Non- structural 1 (NS1) inside arthropod exosomes confirmed that tick exosomes contain viral RNA and proteins. Viral RNA and proteins in exosomes derived from tick and mammalian cells were secured, highly infectious and replicative in all tested evaluations. Furthermore, treatment of tick cells with GW4869, a selective inhibitor that blocks exosome production affected LGTV loads and transmission from arthropod to human cells. 1-D gel electrophoresis further revealed presence of several arthropod exosome-enriched tick molecules. These preliminary results form the strong basis for the proposal to characterize role of arthropod exosomes in tick-LGTV interactions. Several approaches that delineate molecular signaling and identification of arthropod exosomal proteins in tick-LGTV interactions are proposed. We hypothesize that tick exosomal-enriched proteins could be considered as ideal candidates for the development of anti- vector vaccines. The proposed aims provide important insights to define molecular basis of the relationship between tick exosomes and pathogens. This is a transformative and a novel study that not only provides information on the role of arthropod exosomes in vector-pathogen interactions but also lead to the development of better strategies to treat or control transmission of pathogens from this and perhaps other vectors of medical importance.